RESUMEN
Astroglial networks closely interact with neuronal populations, but their functional contribution to neuronal representation of sensory information remains unexplored. The superior colliculus (SC) integrates multi-sensory information by generating distinct spatial patterns of neuronal functional responses to specific sensory stimulation. Here, we report that astrocytes from the mouse SC form extensive networks in the retinorecipient layer compared to visual cortex. This strong astroglial connectivity relies on high expression of gap-junction proteins. Genetic disruption of this connectivity functionally impairs SC retinotopic and orientation preference responses. These alterations are region specific, absent in primary visual cortex, and associated at the circuit level with a specific impairment of collicular neurons synaptic transmission. This has implications for SC-related visually induced innate behavior, as disrupting astroglial networks impairs light-evoked temporary arrest. Our results indicate that astroglial networks shape synaptic circuit activity underlying SC functional visual responses and play a crucial role in integrating visual cues to drive sensory-motor behavior.
Asunto(s)
Astrocitos , Neuronas , Colículos Superiores , Animales , Astrocitos/metabolismo , Astrocitos/fisiología , Ratones , Colículos Superiores/fisiología , Neuronas/fisiología , Neuronas/metabolismo , Ratones Endogámicos C57BL , Transmisión Sináptica/fisiología , Estimulación Luminosa , Corteza Visual/fisiología , Red Nerviosa/fisiología , MasculinoRESUMEN
During brain maturation, astrocytes establish complex morphologies unveiling intense structural plasticity. Connexin 30 (Cx30), a gap-junction channel-forming protein expressed postnatally, dynamically regulates during development astrocyte morphological properties by controlling ramification and extension of fine processes. However, the underlying mechanisms remain unexplored. Here, we found in vitro that Cx30 interacts with the actin cytoskeleton in astrocytes and inhibits its structural reorganization and dynamics during cell migration. This translates into an alteration of local physical surface properties, as assessed by correlative imaging using stimulated emission depletion (STED) super resolution imaging and atomic force microscopy (AFM). Specifically, Cx30 impaired astrocyte cell surface topology and cortical stiffness in motile astrocytes. As Cx30 alters actin organization, dynamics, and membrane physical properties, we assessed whether it controls astrocyte migration. We found that Cx30 reduced persistence and directionality of migrating astrocytes. Altogether, these data reveal Cx30 as a brake for astrocyte structural and mechanical plasticity.
Asunto(s)
Citoesqueleto de Actina , Astrocitos , Movimiento Celular , Conexina 30 , Astrocitos/metabolismo , Animales , Movimiento Celular/fisiología , Citoesqueleto de Actina/metabolismo , Conexina 30/metabolismo , Células Cultivadas , Ratones , Microscopía de Fuerza Atómica/métodos , Ratones Endogámicos C57BLRESUMEN
Fragile X syndrome (FXS) is an inherited form of intellectual disability caused by the loss of the mRNA-binding fragile X mental retardation protein (FMRP). FXS is characterized by neuronal hyperexcitability and behavioral defects, however the mechanisms underlying these critical dysfunctions remain unclear. Here, using male Fmr1 knockout mouse model of FXS, we identify abnormal extracellular potassium homeostasis, along with impaired potassium channel Kir4.1 expression and function in astrocytes. Further, we reveal that Kir4.1 mRNA is a binding target of FMRP. Finally, we show that the deficit in astroglial Kir4.1 underlies neuronal hyperexcitability and several behavioral defects in Fmr1 knockout mice. Viral delivery of Kir4.1 channels specifically to hippocampal astrocytes from Fmr1 knockout mice indeed rescues normal astrocyte potassium uptake, neuronal excitability, and cognitive and social performance. Our findings uncover an important role for astrocyte dysfunction in the pathophysiology of FXS, and identify Kir4.1 channel as a potential therapeutic target for FXS.
Asunto(s)
Astrocitos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil , Neuronas , Canales de Potasio de Rectificación Interna , Animales , Masculino , Ratones , Astrocitos/metabolismo , Conducta Animal , Modelos Animales de Enfermedad , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/fisiopatología , Hipocampo/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Neuronas/fisiología , Potasio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio de Rectificación Interna/genética , ARN Mensajero/metabolismo , ARN Mensajero/genéticaRESUMEN
Connexin 43, an astroglial gap junction protein, is enriched in perisynaptic astroglial processes and plays major roles in synaptic transmission. We have previously found that astroglial Cx43 controls synaptic glutamate levels and allows for activity-dependent glutamine release to sustain physiological synaptic transmissions and cognitiogns. However, whether Cx43 is important for the release of synaptic vesicles, which is a critical component of synaptic efficacy, remains unanswered. Here, using transgenic mice with a glial conditional knockout of Cx43 (Cx43-/-), we investigate whether and how astrocytes regulate the release of synaptic vesicles from hippocampal synapses. We report that CA1 pyramidal neurons and their synapses develop normally in the absence of astroglial Cx43. However, a significant impairment in synaptic vesicle distribution and release dynamics were observed. In particular, the FM1-43 assays performed using two-photon live imaging and combined with multi-electrode array stimulation in acute hippocampal slices, revealed a slower rate of synaptic vesicle release in Cx43-/- mice. Furthermore, paired-pulse recordings showed that synaptic vesicle release probability was also reduced and is dependent on glutamine supply via Cx43 hemichannel (HC). Taken together, we have uncovered a role for Cx43 in regulating presynaptic functions by controlling the rate and probability of synaptic vesicle release. Our findings further highlight the significance of astroglial Cx43 in synaptic transmission and efficacy.
Asunto(s)
Conexina 43 , Vesículas Sinápticas , Ratones , Animales , Conexina 43/metabolismo , Vesículas Sinápticas/metabolismo , Astrocitos/metabolismo , Glutamina/metabolismo , Sinapsis/metabolismo , Hipocampo/metabolismo , Ratones TransgénicosRESUMEN
Astrocytes crucially contribute to synaptic physiology and information processing. One of their key characteristics is to express high levels of connexins (Cxs), the gap junction-forming protein. Among them, Cx30 displays specific properties since it is postnatally expressed and dynamically upregulated by neuronal activity and modulates cognitive processes by shaping synaptic and network activities, as recently shown in knockout mice. However, it remains unknown whether local and selective upregulation of Cx30 in postnatal astrocytes within a physiological range modulates neuronal activities in the hippocampus. We here show in mice that, whereas Cx30 upregulation increases the connectivity of astroglial networks, it decreases spontaneous and evoked synaptic transmission. This effect results from a reduced neuronal excitability and translates into an alteration in the induction of synaptic plasticity and an in vivo impairment in learning processes. Altogether, these results suggest that astroglial networks have a physiologically optimized size to appropriately regulate neuronal functions.
Asunto(s)
Astrocitos , Conexina 43 , Ratones , Animales , Conexina 30/metabolismo , Astrocitos/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Regulación hacia Arriba , Conexinas/genética , Conexinas/metabolismo , Ratones Noqueados , Hipocampo/metabolismoRESUMEN
Astroglial release of molecules is thought to actively modulate neuronal activity, but the nature, release pathway, and cellular targets of these neuroactive molecules are still unclear. Pannexin 1, expressed by neurons and astrocytes, form nonselective large pore channels that mediate extracellular exchange of molecules. The functional relevance of these channels has been mostly studied in brain tissues, without considering their specific role in different cell types, or in neurons. Thus, our knowledge of astroglial pannexin 1 regulation and its control of neuronal activity remains very limited, largely due to the lack of tools targeting these channels in a cell-specific way. We here show that astroglial pannexin 1 expression in mice is developmentally regulated and that its activation is activity-dependent. Using astrocyte-specific molecular tools, we found that astroglial-specific pannexin 1 channel activation, in contrast to pannexin 1 activation in all cell types, selectively and negatively regulates hippocampal networks, with their disruption inducing a drastic switch from bursts to paroxysmal activity. This decrease in neuronal excitability occurs via an unconventional astroglial mechanism whereby pannexin 1 channel activity drives purinergic signaling-mediated regulation of hyperpolarisation-activated cyclic nucleotide (HCN)-gated channels. Our findings suggest that astroglial pannexin 1 channel activation serves as a negative feedback mechanism crucial for the inhibition of hippocampal neuronal networks.
Asunto(s)
Astrocitos , Conexinas , Modelos Animales de Enfermedad , Animales , Ratones , Conexinas/metabolismo , Astrocitos/metabolismoRESUMEN
Presynaptic glutamate replenishment is fundamental to brain function. In high activity regimes, such as epileptic episodes, this process is thought to rely on the glutamate-glutamine cycle between neurons and astrocytes. However the presence of an astroglial glutamine supply, as well as its functional relevance in vivo in the healthy brain remain controversial, partly due to a lack of tools that can directly examine glutamine transfer. Here, we generated a fluorescent probe that tracks glutamine in live cells, which provides direct visual evidence of an activity-dependent glutamine supply from astroglial networks to presynaptic structures under physiological conditions. This mobilization is mediated by connexin43, an astroglial protein with both gap-junction and hemichannel functions, and is essential for synaptic transmission and object recognition memory. Our findings uncover an indispensable recruitment of astroglial glutamine in physiological synaptic activity and memory via an unconventional pathway, thus providing an astrocyte basis for cognitive processes.
Asunto(s)
Astrocitos/metabolismo , Glutamina/metabolismo , Hipocampo/fisiología , Reconocimiento en Psicología , Transmisión Sináptica , Animales , Cognición , Colorantes Fluorescentes/química , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Glutamina/química , Hipocampo/citología , Microscopía Intravital , Masculino , Ratones , Ratones Transgénicos , Modelos Animales , Sondas Moleculares , Neuronas/metabolismo , Rodaminas/química , Técnicas EstereotáxicasRESUMEN
Brain postnatal development is characterized by critical periods of experience-dependent remodeling of neuronal circuits. Failure to end these periods results in neurodevelopmental disorders. The cellular processes defining critical-period timing remain unclear. Here, we show that in the mouse visual cortex, astrocytes control critical-period closure. We uncover the underlying pathway, which involves astrocytic regulation of the extracellular matrix, allowing interneuron maturation. Unconventional astrocyte connexin signaling hinders expression of extracellular matrix-degrading enzyme matrix metalloproteinase 9 (MMP9) through RhoA-guanosine triphosphatase activation. Thus, astrocytes not only influence the activity of single synapses but also are key elements in the experience-dependent wiring of brain circuits.
Asunto(s)
Astrocitos/fisiología , Período Crítico Psicológico , Plasticidad Neuronal , Corteza Visual/crecimiento & desarrollo , Animales , Astrocitos/metabolismo , Conexina 30/metabolismo , Activación Enzimática , GTP Fosfohidrolasas/metabolismo , Interneuronas/metabolismo , Interneuronas/fisiología , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Sinapsis/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The water channel protein aquaporin-4 (AQP4) and the gap junction forming proteins connexin-43 (Cx43) and connexin-30 (Cx30) are astrocytic proteins critically involved in brain water and ion homeostasis. While AQP4 is mainly involved in water flux across the astrocytic endfeet membranes, astrocytic gap junctions provide syncytial coupling allowing intercellular exchange of water, ions, and other molecules. We have previously shown that mice with targeted deletion of Aqp4 display enhanced gap junctional coupling between astrocytes. Here, we investigate whether uncoupling of the astrocytic syncytium by deletion of the astrocytic connexins Cx43 and Cx30 affects AQP4 membrane localization and expression. By using quantitative immunogold cytochemistry, we show that deletion of astrocytic connexins leads to a substantial reduction of perivascular AQP4, concomitant with a down-regulation of total AQP4 protein and mRNA. Isoform expression analysis shows that while the level of the predominant AQP4 M23 isoform is reduced in Cx43/Cx30 double deficient hippocampal astrocytes, the levels of M1, and the alternative translation AQP4ex isoform protein levels are increased. These findings reveal a complex interdependence between AQP4 and connexins, which are both significantly involved in homeostatic functions and astrogliopathologies.
Asunto(s)
Acuaporina 4/metabolismo , Astrocitos/metabolismo , Células Gigantes/metabolismo , Animales , Acuaporina 4/genética , Proteínas de Unión al Calcio/metabolismo , Conexinas/metabolismo , Eliminación de Gen , Hipocampo/metabolismo , Hipocampo/ultraestructura , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Proteínas Musculares/metabolismo , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
As the most abundant gap junction protein in the central nervous system (CNS), astrocytic connexin 43 (Cx43) maintains astrocyte network homeostasis, affects oligodendroglial development and participates in CNS pathologies as well as injury progression. However, its role in remyelination is not yet fully understood. To address this issue, we used astrocyte-specific Cx43 conditional knockout (Cx43 cKO) mice generated through the use of a hGFAP-cre promoter, in combination with mice carrying a floxed Cx43 allele that were subjected to lysolecithin so as to induce demyelination. We found no significant difference in the demyelination of the corpus callosum between Cx43 cKO mice and their non-cre littermate controls, while the remyelination process in Cx43 cKO mice was accelerated. Moreover, an increased number of mature oligodendrocytes and an unaltered number of oligodendroglial lineage cells were found in Cx43 cKO mouse lesions. This indicates that oligodendrocyte precursor cell (OPC) differentiation was facilitated by astroglial Cx43 depletion as remyelination progressed. Underlying the latter, there was a down-regulated glial activation and modulated local inflammation as well as a reduction of myelin debris in Cx43 cKO mice. Importantly, 2 weeks of orally administrating boldine, a natural alkaloid that blocks Cx hemichannel activity in astrocytes without affecting gap junctional communication, obviously modulated local inflammation and promoted remyelination. Together, the data suggest that the astrocytic Cx43 hemichannel is negatively involved in the remyelination process by favoring local inflammation. Consequently, inhibiting Cx43 hemichannel functionality may be a potential therapeutic approach for demyelinating diseases in the CNS.
Asunto(s)
Astrocitos/metabolismo , Conexina 43/metabolismo , Inflamación/metabolismo , Remielinización/fisiología , Animales , Diferenciación Celular/fisiología , Sistema Nervioso Central/metabolismo , Enfermedades Desmielinizantes/patología , Uniones Comunicantes/metabolismo , Ratones , Vaina de Mielina/metabolismo , Células Precursoras de Oligodendrocitos/metabolismo , Oligodendroglía/metabolismoRESUMEN
Astrocytes play key roles in brain functions through dynamic interactions with neurons. One of their typical features is to express high levels of connexins (Cxs), Cx43 and Cx30, the gap junction (GJ)-forming proteins. Cx30 is involved in basic cognitive processes and shapes synaptic and network activities, as shown by recent studies in transgenic animals. Yet it remains unknown whether astroglial Cx30 expression, localization, and functions are endogenously and dynamically regulated by neuronal activity and could therefore play physiological roles in neurotransmission. We here show that neuronal activity increased hippocampal Cx30 protein levels via a posttranslational mechanism regulating lysosomal degradation. Neuronal activity also increased Cx30 protein levels at membranes and perisynaptic processes, as revealed by superresolution imaging. This translated at the functional level in the activation of Cx30 hemichannels and in Cx30-mediated remodeling of astrocyte morphology independently of GJ biochemical coupling. Altogether, these data show activity-dependent dynamics of Cx30 expression, perisynaptic localization, and functions.
Asunto(s)
Astrocitos/fisiología , Conexina 30/fisiología , Hipocampo/fisiología , Neuronas/fisiología , Animales , Astrocitos/citología , Femenino , Hipocampo/citología , Lisosomas/fisiología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , ProteolisisRESUMEN
Astroglial networks mediated by gap junction channels contribute to neurotransmission and promote neuronal coordination. Connexin 30, one of the two main astroglial gap junction forming protein, alters at the behavioral level the reactivity of mice to novel environment and at the synaptic level excitatory transmission. However, the role and function of Cx30 at the neuronal network level remain unclear. We thus investigated whether Cx30 regulates neuronal population bursts and associated convulsive behavior. We found in vivo that Cx30 is upregulated by kainate-induced seizures and that it regulates in turn the severity of associated behavioral seizures. Using electrophysiology ex vivo, we report that Cx30 regulates aberrant network activity via control of astroglial glutamate clearance independently of gap-junction mediated biochemical coupling. Altogether, our results indicate that astroglial Cx30 is an important player in orchestrating neuronal network activity.
Asunto(s)
Astrocitos/metabolismo , Conexina 30/biosíntesis , Uniones Comunicantes/metabolismo , Red Nerviosa/metabolismo , Neuronas/metabolismo , Animales , Astrocitos/química , Conexina 30/análisis , Femenino , Uniones Comunicantes/química , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Red Nerviosa/química , Neuronas/químicaRESUMEN
The precise contribution of astrocytes in neuroinflammatory process occurring in Parkinson's disease (PD) is not well characterized. In this study, using GRCx30CreERT2 mice that are conditionally inactivated for glucocorticoid receptor (GR) in astrocytes, we have examined the actions of astrocytic GR during dopamine neuron (DN) degeneration triggered by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results show significantly augmented DN loss in GRCx30CreERT2 mutant mice in substantia nigra (SN) compared to controls. Hypertrophy of microglia but not of astrocytes was greatly enhanced in SN of these astrocytic GR mutants intoxicated with MPTP, indicating heightened microglial reactivity compared to similarly-treated control mice. In the SN of GR astrocyte mutants, specific inflammation-associated transcripts ICAM-1, TNF-α and Il-1ß as well as TNF-α protein levels were significantly elevated after MPTP neurotoxicity compared to controls. Interestingly, this paralleled increased connexin hemichannel activity and elevated intracellular calcium levels in astrocytes examined in acute midbrain slices from control and mutant mice treated with MPP+ . The increased connexin-43 hemichannel activity was found in vivo in MPTP-intoxicated mice. Importantly, treatment of MPTP-injected GRCx30CreERT2 mutant mice with TAT-Gap19 peptide, a specific connexin-43 hemichannel blocker, reverted both DN loss and microglial activation; in wild-type mice there was partial but significant survival effect. In the SN of post-mortem PD patients, a significant decrease in the number of astrocytes expressing nuclear GR was observed, suggesting the participation of astrocytic GR deregulation of inflammatory process in PD. Overall, these data provide mechanistic insights into GR-modulated processes in vivo, specifically in astrocytes, that contribute to a pro-inflammatory state and dopamine neurodegeneration in PD pathology.
Asunto(s)
Astrocitos/metabolismo , Conexinas/metabolismo , Neuronas Dopaminérgicas/metabolismo , Enfermedad de Parkinson/genética , Animales , Humanos , Masculino , Ratones , Enfermedad de Parkinson/patologíaRESUMEN
Astrocytes undergo intense morphological maturation during development, changing from individual sparsely branched cells to polarized and tremendously ramified cells. Connexin 30, an astroglial gap-junction channel-forming protein expressed postnatally, regulates in situ the extension and ramification of astroglial processes. However, the involvement of connexin 30 in astroglial polarization, which is known to control cell morphology, remains unexplored. We found that connexin 30, independently of gap-junction-mediated intercellular biochemical coupling, alters the orientation of astrocyte protrusion, centrosome and Golgi apparatus during polarized migration in an in vitro wound-healing assay. Connexin 30 sets the orientation of astroglial motile protrusions via modulation of the laminin/ß1 integrin/Cdc42 polarity pathway. Connexin 30 indeed reduces laminin levels, inhibits the redistribution of the ß1-integrin extracellular matrix receptors, and inhibits the recruitment and activation of the small Rho GTPase Cdc42 at the leading edge of migrating astrocytes. In vivo, connexin 30, the expression of which is developmentally regulated, also contributes to the establishment of hippocampal astrocyte polarity during postnatal maturation. This study thus reveals that connexin 30 controls astroglial polarity during development.
Asunto(s)
Astrocitos/citología , Encéfalo/citología , Polaridad Celular/fisiología , Conexina 30/metabolismo , Animales , Astrocitos/fisiología , Encéfalo/metabolismo , Encéfalo/fisiología , Ensayos de Migración Celular , Técnica del Anticuerpo Fluorescente , RatonesRESUMEN
Astrocytes interact dynamically with neurons by modifying synaptic activity and plasticity. This interplay occurs through a process named gliotransmission, meaning that neuroactive molecules are released by astrocytes. Acting as a gliotransmitter, D-serine, a co-agonist of the NMDA receptor at the glycine-binding site, can be released by astrocytes in a calcium [Ca2+]i-dependent manner. A typical feature of astrocytes is their high expression level of connexin43 (Cx43), a protein forming gap junction channels and hemichannels associated with dynamic neuroglial interactions. Pharmacological and genetic inhibition of Cx43 hemichannel activity reduced the amplitude of NMDA EPSCs in mouse layer 5 prefrontal cortex pyramidal neurons without affecting AMPA EPSC currents. This reduction of NMDA EPSCs was rescued by addition of D-serine in the extracellular medium. LTP of NMDA and AMPA EPSCs after high-frequency stimulation was reduced by prior inhibition of Cx43 hemichannel activity. Inactivation of D-serine synthesis within the astroglial network resulted in the reduction of NMDA EPSCs, which was rescued by adding extracellular D-serine. We showed that the activity of Cx43 hemichannels recorded in cultured astrocytes was [Ca2+]I dependent. Accordingly, in acute cortical slices, clamping [Ca2+]i at a low level in astroglial network resulted in an inhibition of NMDA EPSC potentiation that was rescued by adding extracellular D-serine. This work demonstrates that astroglial Cx43 hemichannel activity is associated with D-serine release. This process, occurring by direct permeation of D-serine through hemichannels or indirectly by Ca2+ entry and activation of other [Ca2+]i-dependent mechanisms results in the modulation of synaptic activity and plasticity.SIGNIFICANCE STATEMENT We recorded neuronal glutamatergic (NMDA and AMPA) responses in prefrontal cortex (PFC) neurons and used pharmacological and genetic interventions to block connexin-mediated hemichannel activity specifically in a glial cell population. For the first time in astrocytes, we demonstrated that hemichannel activity depends on the intracellular calcium concentration and is associated with D-serine release. Blocking hemichannel activity reduced the LTP of these excitatory synaptic currents triggered by high-frequency stimulation. These observations may be particularly relevant in the PFC, where D-serine and its converting enzyme are highly expressed.
Asunto(s)
Astrocitos/fisiología , Señalización del Calcio/fisiología , Conexina 43/metabolismo , Ácido Glutámico/metabolismo , Corteza Prefrontal/fisiología , Serina/metabolismo , Transmisión Sináptica/fisiología , Animales , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Plasticidad Neuronal/fisiología , Neurotransmisores/metabolismoRESUMEN
The contribution of reactive gliosis to the pathological phenotype of Alzheimer's disease (AD) opened the way for therapeutic strategies targeting glial cells instead of neurons. In such context, connexin hemichannels were proposed recently as potential targets since neuronal suffering is alleviated when connexin expression is genetically suppressed in astrocytes of a murine model of AD. Here, we show that boldine, an alkaloid from the boldo tree, inhibited hemichannel activity in astrocytes and microglia without affecting gap junctional communication in culture and acute hippocampal slices. Long-term oral administration of boldine in AD mice prevented the increase in glial hemichannel activity, astrocytic Ca2+ signal, ATP and glutamate release and alleviated hippocampal neuronal suffering. These findings highlight the important pathological role of hemichannels in AD mice. The neuroprotective effect of boldine treatment might provide the basis for future pharmacological strategies that target glial hemichannels to reduce neuronal damage in neurodegenerative diseases.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Aporfinas/farmacología , Aporfinas/uso terapéutico , Conexinas/metabolismo , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Células Cultivadas , Conexinas/genética , Modelos Animales de Enfermedad , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Hipocampo/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuritas/metabolismo , Neuritas/patología , Neuroglía/metabolismo , Fármacos Neuromusculares Despolarizantes/farmacología , Fármacos Neuromusculares Despolarizantes/uso terapéutico , Neuronas/fisiología , Neurotransmisores/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismoRESUMEN
The non-receptor tyrosine kinase c-Src is an important mediator in several signaling pathways related to neuroinflammation. Our previous study showed that cortical injection of kainic acid (KA) promoted a transient increase in c-Src activity in reactive astrocytes surrounding the neuronal lesion. As a cell-penetrating peptide based on connexin43 (Cx43), specifically TAT-Cx43266-283, inhibits Src activity, we investigated the effect of TAT-Cx43266-283 on neuronal death promoted by cortical KA injections in adult mice. As expected, KA promoted neuronal death, estimated by the reduction in NeuN-positive cells and reactive gliosis, characterized by the increase in glial fibrillary acidic protein (GFAP) expression. Interestingly, TAT-Cx43266-283 injected with KA diminished neuronal death and reactive gliosis compared to KA or KA+TAT injections. In order to gain insight into the neuroprotective mechanism, we used in vitro models. In primary cultured neurons, TAT-Cx43266-283 did not prevent neuronal death promoted by KA, but when neurons were grown on top of astrocytes, TAT-Cx43266-283 prevented neuronal death promoted by KA. These observations demonstrate the participation of astrocytes in the neuroprotective effect of TAT-Cx43266-283. Furthermore, the neuroprotective effect was also present in non-contact co-cultures, suggesting the contribution of soluble factors released by astrocytes. As glial hemichannel activity is associated with the release of several factors, such as ATP and glutamate, that cause neuronal death, we explored the participation of these channels on the neuroprotective effect of TAT-Cx43266-283. Our results confirmed that inhibitors of ATP and NMDA receptors prevented neuronal death in co-cultures treated with KA, suggesting the participation of astrocyte hemichannels in neurotoxicity. Furthermore, TAT-Cx43266-283 reduced hemichannel activity promoted by KA in neuron-astrocyte co-cultures as assessed by ethidium bromide (EtBr) uptake assay. In fact, TAT-Cx43266-283 and dasatinib, a potent c-Src inhibitor, strongly reduced the activation of astrocyte hemichannels. In conclusion, our results suggest that TAT-Cx43266-283 exerts a neuroprotective effect through the reduction of hemichannel activity likely mediated by c-Src in astrocytes. These data unveil a new role of c-Src in the regulation of Cx43-hemichannel activity that could be part of the mechanism by which astroglial c-Src participates in neuroinflammation.
RESUMEN
Antidepressants, prescribed as first line treatment of neuropathic pain, have a limited efficacy and poorly tolerated side effects. Because recent studies pointed out the implication of astroglial connexins (Cx) in both neuropathic pain and antidepressive treatment, we investigated whether their blockade by mefloquine could modulate the action of the tricyclic antidepressant amitriptyline. Using primary cultures, we found that both mefloquine and amitriptyline inhibited Cx43-containing gap junctions, and that the drug combination acted synergically. We then investigated whether mefloquine could enhance amitriptyline efficacy in a preclinical model of neuropathic pain. Sprague-Dawley rats that underwent chronic unilateral constriction injury (CCI) to the sciatic nerve (SN) were treated with either amitriptyline, mefloquine or the combination of both drugs. Whereas acute treatments were ineffective, chronic administration of amitriptyline reduced CCI-SN-induced hyperalgesia-like behavior, and this effect was markedly enhanced by co-administration of mefloquine, which was inactive on its own. No pharmacokinetic interactions between both drugs were observed and CCI-SN-induced neuroinflammatory and glial activation markers remained unaffected by these treatments in dorsal root ganglia and spinal cord. Mechanisms downstream of CCI-SN-induced neuroinflammation and glial activation might therefore be targeted. Connexin inhibition in astroglia could represent a promising approach towards improving neuropathic pain therapy by antidepressants.
Asunto(s)
Amitriptilina/farmacología , Astrocitos/metabolismo , Conexina 43/antagonistas & inhibidores , Uniones Comunicantes/metabolismo , Hiperalgesia/tratamiento farmacológico , Neuralgia/tratamiento farmacológico , Neuropatía Ciática/tratamiento farmacológico , Animales , Astrocitos/patología , Conexina 43/metabolismo , Uniones Comunicantes/patología , Hiperalgesia/metabolismo , Hiperalgesia/patología , Masculino , Neuralgia/metabolismo , Neuralgia/patología , Ratas , Ratas Sprague-Dawley , Neuropatía Ciática/metabolismo , Neuropatía Ciática/patologíaRESUMEN
The main energy source powering the brain is glucose. Strong energy needs of our nervous system are fulfilled by conveying this essential metabolite through blood via an extensive vascular network. Glucose then reaches brain tissues by cell uptake, diffusion and metabolization, processes primarily undertaken by astrocytes. Deprivation of glucose can however occur in various circumstances. In particular, ageing is associated with cognitive disturbances that are partly attributable to metabolic deficiency leading to brain glycopenia. Despite the crucial role of glucose and its metabolites in sustaining neuronal activity, little is known about its moment-to-moment contribution to astroglial physiology. We thus here investigated the early structural and functional alterations induced in astrocytes by a transient metabolic challenge consisting in glucose deprivation. Electrophysiological recordings of hippocampal astroglial cells of the stratum radiatum in situ revealed that shortage of glucose specifically increases astrocyte membrane capacitance, whilst it has no impact on other passive membrane properties. Consistent with this change, morphometric analysis unraveled a prompt increase in astrocyte volume upon glucose deprivation. Furthermore, characteristic functional properties of astrocytes are also affected by transient glucose deficiency. We indeed found that glucoprivation decreases their gap junction-mediated coupling, while it progressively and reversibly increases their intracellular calcium levels during the slow depression of synaptic transmission occurring simultaneously, as assessed by dual electrophysiological and calcium imaging recordings. Together, these data indicate that astrocytes rapidly respond to metabolic dysfunctions, and are therefore central to the neuroglial dialog at play in brain adaptation to glycopenia.
RESUMEN
In the normal brain, immune cell trafficking and immune responses are strictly controlled and limited. This unique homeostatic equilibrium, also called brain immune quiescence, is crucial to maintaining proper brain functions and is altered in various pathological processes, from chronic immunopathological disorders to cognitive and psychiatric impairments. To date, the precise nature of factors regulating the brain/immune system interrelationship is poorly understood. In the present study, we demonstrate that one of these regulating factors is Connexin 43 (Cx43), a gap junction protein highly expressed by astrocytes at the blood-brain barrier (BBB) interface. We show that, by setting the activated state of cerebral endothelium, astroglial Cx43 controls immune recruitment as well as antigen presentation mechanisms in the mouse brain. Consequently, in the absence of astroglial Cx43, recruited immune cells elaborate a specific humoral autoimmune response against the von Willebrand factor A domain-containing protein 5a, an extracellular matrix protein of the brain. Altogether, our results demonstrate that Cx43 is a new astroglial factor promoting the immune quiescence of the brain.